From the Department of Pharmacology III (H.K., M.O., K.K., Y.F.),
Discovery Research Laboratories; the Research and Development Coordination
(A.N.) and the Department of Toxicology (K.T.), Developmental Research
Laboratories, Dainippon Pharmaceutical Co., Osaka; and the Department of
Metabolic Diseases (H.K., S.K., T.K.), Graduate School of Medicine, University
of Tokyo, Tokyo, Japan.

Abstract

The mechanism by which the specific β3-adrenoceptor agonist
AJ-9677 relieves insulin resistance in vivo was investigated by studying its
effects in the white and brown adipose tissues of the
KK-Ay/Ta diabetic obese mouse model. AJ-9677 reduced the
total weight of white adipose tissues by reducing the size of the adipocytes,
an effect associated with the normalization of tumor necrosis factor-α
(TNF-α) and leptin expression levels. The levels of uncoupling protein
(UCP)-1 mRNA in brown adipose tissue were increased threefold. AJ-9677 caused
a marked increase (20- to 80-fold) in the expression of UCP-1 in white adipose
tissues. The levels of UCP-2 mRNA were increased in both the white and brown
adipose tissues of diabetic obese mice, and AJ-9677 further upregulated UCP-2
mRNA levels in brown adipose tissue, but reduced its levels in white adipose
tissue. UCP-3 mRNA levels were not essentially changed by AJ-9677. However,
AJ-9677 significantly (two- to four-fold) upregulated the GLUT4 mRNA and
protein levels in white and brown adipose tissues and the gastrocnemius. The
generation of small adipocytes, presumably mediated by increased expression of
UCP-1 in addition to increased lipolysis in response to AJ-9677, was
associated with decreased TNF-α and free fatty acid production and may
be the mechanism of amelioration of insulin resistance in
KK-Ay/Ta diabetic obese mice.

The β3-adrenoceptor was suspected to exist more than 15
years ago (1), and it is now
known to be present in both rodent and human tissues. The human
β3-adrenoceptor was cloned and sequenced in 1989
(2). The characteristics of the
β3-adrenoceptor are quite different from those of
β1- and β2-adrenoceptors. The
β3-adrenoceptor is expressed mainly in the white and brown
adipose tissues (3), and it is
important in lipolysis and thermogenesis in rodents
(4). The
β3-adrenoceptor agonist BRL 26830A stimulates lipolysis in
adipose tissues, and chronic treatment with an adequate dose of BRL 26830A has
decreased body weight without reducing food intake
(5). This weight loss was
caused by the stimulation of energy expenditure and increased lipolysis
(6). The effects of the
β3-adrenoceptor on thermogenesis are believed to occur through
activation and upregulation of uncoupling protein (UCP)-1, which is mainly
expressed in brown adipose tissues. For example, chronic treatment of yellow
KK mice with the β3-adrenoceptor agonist CL 316,243 increased
expression of UCP-1 mRNA in brown adipose tissues and induced UCP-1 mRNA in
white adipose tissues, the gastrocnemius, and quadriceps muscles
(7). Recently, UCP-2 and -3
were identified. UCP-2 is widely expressed in humans; UCP-2 mRNA has been
detected in the adipose tissues, skeletal muscles, lungs, the heart, and
kidneys
(8,9).
UCP-3 is expressed primarily in skeletal muscles in humans and in skeletal
muscles and brown adipose tissues in rodents
(10,11).
Regulation of the activation of UCP-2 and -3 may be different from that of
UCP-1
(10,12,13),
and these two proteins may be functionally involved in energy expenditure
(8,10).
Therefore, the increased energy expenditure induced by
β3-adrenoceptor agonists in vivo may be mediated by UCP-2 and
-3 in addition to UCP-1, especially in humans.

β3-adrenoceptor agonists have antidiabetic and antiobesity
effects in rodent models of type 2 diabetes
(14,15,16,17),
but the molecular mechanisms of these effects, especially the antidiabetic
effects, are largely unknown. Molecules secreted by hypertrophic adipocytes
(e.g., tumor necrosis factor-α [TNF-α] and free fatty acids
[FFAs]) may cause insulin resistance associated with obese type 2 diabetes.
The expression levels of TNF-α in white adipose tissues are higher in
diabetic obese models than in normal animals, and both neutralization of
TNF-α (18) and target
disruption of the TNF-α/TNF-α receptor
(19) have prevented the
development of insulin resistance despite the presence of obesity. TNF-α
appears to interfere with the insulin signal transduction pathway by
inhibiting the insulin receptor tyrosine kinase and the tyrosine
phosphorylation of insulin receptor substrate-1
(18). Moreover, FFAs cause
insulin resistance in the skeletal muscles and liver via multiple mechanisms,
including the inhibition of phosphatidylinositol 3-kinase activity associated
with insulin receptor substrate-1
(20), which may cause
inhibition of GLUT4 translocation and glycogen synthesis stimulated by
insulin. The expression levels of leptin in white adipose tissues and the
plasma levels secreted from adipocytes are higher in diabetic obese models.
Administration of leptin causes insulin sensitivity rather than insulin
resistance (21). Leptin may be
secreted from hypertrophic adipocytes to compensate for the insulin resistance
caused by obesity. In fact, some methods used to cause insulin resistance
(e.g., lipid infusion and glucose infusion) were recently found to stimulate
leptin gene expression (22).
Thiazolidinediones improve insulin sensitivity in diabetic obese models. They
increase the number of small white adipocytes and decrease the number of large
adipocytes, thereby reducing expression levels of TNF-α and plasma FFA
concentrations (23). The
regulation of TNF-α expression and FFA production in white adipose
tissues may be closely involved in the antidiabetic effect of
β3-adrenoceptor agonists.

Many β3-adrenoceptor agonists have been developed, but the
early compounds did not show the expected effects in humans that were seen in
mice and rats. After the cloning of the human β3-adrenoceptor,
differences between the structures of the rat and human receptors were
clarified. AJ-9677 was screened with Chinese hamster ovary cells expressing
either the human or the rat β3-adrenoceptor
(24). We previously
demonstrated that AJ-9677 could stimulate both rat and human
β3-adrenoceptors
(24). The present study
investigated the effects on diabetes and obesity and the molecular mechanisms
of AJ-9677 in the KK-Ay/Ta diabetic obese mouse model.

Animals and drug treatment. Male 9-week-old KK-Ay/Ta and 11-week-old C57BL/6J mice were purchased from Clea Japan (Tokyo, Japan) and were allowed to adjust to the facilities and handling for 1 week before all experimental protocols. Under standardized conditions, the mice were given dry food (CE-2; Oriental Yeast, Tokyo) and water ad libitum. Mice were divided into control and treatment groups so that the mean body weight of the two groups was comparable 1 day before the starting day (day 1). The treatment group was given AJ-9677 by gavage at 0.01 mg/kg or 0.1
mg-1·kg-1·day-1 in the morning
for 14 days. The control group was given only 0.5% tragacanth solution. At 24
h after the last administration of AJ-9677, blood samples were collected from
the cut ends of the tails and centrifuged to separate plasma. Blood samples
from 12-week-old C57BL/6J mice were also collected by the same method. For
some mice, the rectal temperature was measured after AJ-9677 administration.
Some mice were killed by bleeding under anesthesia 20 h after the final
administration of AJ-9677. Epididymal white adipose tissues, inguinal white
adipose tissues, interscapular brown adipose tissues, and the gastrocnemius
were removed and divided into segments for further studies. The same tissues
from 12-week-old C57BL/6J mice were also removed. Some
KK-Ay/Ta mice were administered AJ-9677 (0.1 mg/kg) after
24-h fasting to observe the acute effect on lipolytic activity in vivo. Blood
samples were collected 1 h after the administration.

Oral glucose tolerance test and determination of plasma glucose,
insulin, FFAs, and triglycerides. The oral glucose tolerance test (OGTT)
was performed on day 15 after 24 h fasting. The mice received a 20% glucose
solution (2 g/kg). Blood samples were collected just before and 0.5, 1, 2, and
3 h after glucose loading. Plasma glucose and insulin levels were determined
with a Glucose C-II Test from Wako Pure Chemical Industries (Osaka, Japan) and
an insulin enzyme-linked immunosorbent assay (ELISA) from Shibayagi, (Gumma,
Japan). FFAs were determined with a nonesterified fatty acid (NEFA) C-Test
from Wako Pure Chemical Industries, and Triglycerides were detected with a
Determiner triglyceride-S555 from Kyowa Medex Co. (Tokyo, Japan).

Expression of TNF-α and leptin. Measurements of TNF-α
and leptin proteins secreted in isolated white adipose tissues (400-1,000 mg)
were performed as described previously
(26). Adipose tissues were
minced into small pieces and incubated in a Krebs-Ringer phosphate solution
(pH 7.4) containing endotoxin-free bovine serum albumin (4 g/100 ml) and
glucose (1 mg/ml) at 37°C with rotation (100 rpm). After a 2-h incubation,
the incubation mixture was centrifuged and the supernatant collected.
Concentrations of TNF-α and leptin proteins were determined with a mouse
TNF-α ELISA system (Amersham Pharmacia Biotech U.K., Buckinghamshire,
U.K.) and a mouse leptin ELISA kit (Morinaga Bioscience Institute, Yokohama,
Japan). Total DNA was also isolated from the adipose tissues as described
previously (27).

Histological analysis. Small pieces of epididymal and inguinal white
adipose tissues were removed and rinsed with saline. The tissues were fixed
with 10% formalin and embedded in paraffin. Tissue sections were cut at a
thickness of 2.5 μm and stained with hematoxylin and eosin. To examine the
size of the white adipocytes, the number of adipocytes was counted in five
limited areas (7 × 10-3 mm2) of each stained
specimen. The mean value of the five areas was designated as an index of the
cell size (i.e., a larger number means smaller size). In this analysis, the
multilocular adipocytes were excluded. For immunohistochemistry, the
paraffin-embedded sections were stained with anti-cytochrome oxidase antibody
(Molecular Probes, Eugene, OR). The signals were detected by a Vectastain
avitin-biotin-peroxidase complex system with a diaminobenzidine substrate kit
(Vector Laboratories, Burlingame, CA). For electron microscopic observations,
the tissues were fixed with 2.5% glutaraldehyde and embedded in Epon 812.

Triglycerides and DNA in adipose tissues. The method for determining
the triglyceride and DNA contents in the epididymal and inguinal white adipose
tissues was slightly modified from the method described by Okuno et al.
(23). Briefly, 50 mg adipose
tissue was homogenized in 2 ml of a solution containing 150 mmol/l sodium
chloride, 0.1% Triton X-100, and 10 mmol/l Tris, pH 8.0, at 50°C using a
polytron homogenizer (NS-310E; Micro Tech Nichion, Chiba, Japan) for 1 min.
The triglyceride content of this homogenized solution was determined by a
Determiner triglyceride-S555 (Kyowa Medex). For DNA determination, the
homogenized solution was mixed with SDS, proteinase K, and EDTA to final
concentrations of 0.1%, 100 μg/ml, and 10 mmol/l, respectively. After a
12-16 h incubation at 37°C, the DNA was extracted by the phenol-chloroform
extraction method. The DNA pellets were redissolved in a solution containing
20 μg/ml ribonuclease A, 1 mmol/l EDTA, and 10 mmol/l Tris at pH 8.0. After
20 min of incubation at 37°C, the DNA was re-extracted by the
phenol-chloroform extraction method. The DNA pellets were finally dissolved
with TE buffer (10 mmol/l Tris and 1 mmol/l EDTA, pH 8.0). DNA content was
calculated from the absorbance at 260 nm, with the optical density at 260 nm
of 50 μg/ml DNA solution taken as equal to 1.0 by a Gene Quant RNA/DNA
Calculator (Amersham Pharmacia Biotech, U.K.).

Expression of mRNAs by reverse transcription-competitive polymerase
chain reaction. Extraction of total RNA from tissues was carried out by
homogenizing 50 mg frozen tissue in 1 ml RNAzol B (Tel-Test, Friendswood, TX).
RNA pellets were obtained from the homogenate by repeated extraction with
chloroform and alcohol precipitation. The mRNA of TNF-α, leptin, GLUT4,
and three different subtypes of UCP were quantitatively determined by the
reverse transcription (RT)-competitive polymerase chain reaction (PCR) method
described by Auboeuf and Vidal
(28); however, the internal
standard was prepared according to the method of Celi et al.
(29).
Table 1 lists both the sequences
of primers used for internal standards and RT-competitive PCR and the sizes of
each target fragment and competitor fragment generated by RT-competitive PCR.
For construction of single-strand cDNA, 1 μg of total RNA was used with the
SuperScript preamplification system (Life Technologies, Gaithersburg, MD)
using oligo (dT)12-18 as a primer. The internal standards
(competitors) were prepared by PCR using template cDNA constructed with total
RNA from epididymal white or interscapular brown adipose tissues and a
specific set of oligonucleotides as primers (i.e., forward-short [S] and
reverse) (Table 1).
RT-competitive PCR was performed with a constant amount of target cDNA and
four different amounts of the corresponding internal standard. The PCR
conditions were 20-30 cycles of denaturation at 94°C for 15 s, annealing
at 55°C (or 62°C for TNF-α) for 30 s, and extension at 72°C
for 30 s. The number of cycles was selected to obtain a measurable amount of
products depending on the content of the target. The PCR products were
subjected to agarose gel electrophoresis and ethidium bromide staining. After
photographs were taken, the density of the DNA band was analyzed with the
Kodak Digital Science Electrophoresis Documentation and Analysis System 120
(Eastman Kodak Company, Rochester, NY). The initial amount of the target was
calculated from the plot of the density ratio of the competitor band to the
target band versus the initial amount of the competitor added in the PCR
reaction.

Western blot analysis of GLUT4. Crude and plasma membranes were
prepared from frozen epididymal white adipose tissues using differential
ultracentrifugation as described by Simpson et al.
(36) and Kelada et al.
(37). Proteins in crude and
plasma membrane fractions were separated by SDS-PAGE on 12.5% gels using the
system described by Laemmli
(38). The separated proteins
were transferred to polyvinylidene difluoride (PVDF) membranes at 10 V for 30
min by the semidry blotting system (Bio-Rad Laboratories, Hercules, CA). The
PVDF membranes were blocked with Block Ace (Dainippon Pharmaceutical) and
incubated for 1 h with polyclonal anti-GLUT4 antibody (Transformation
Research, Framingham, MA). The membranes were subsequently incubated with
anti-rabbit IgG antibody conjugated with horseradish peroxidase (Santa Cruz
Biotechnology, Santa Cruz, CA) for 1 h. The immunolabeled signals were
detected with autoradiography film using the enhanced chemiluminescence system
(Amersham Pharmacia Biotech).

Statistical analysis. Values are shown as the means ± SE. The
statistical significance of the values was analyzed by the Dunnett's
two-tailed test when more than three groups were compared or the two-tailed
Student's t test or the Welch t test when two groups were
compared.

Lipolytic activities of AJ-9677 in white adipocytes from
KK-Ay/Ta diabetic obese mice. Isolated adipocytes were
incubated with AJ-9677 (•) or isoproterenol (○). FFA concentrations
were normalized to protein concentrations. The data are expressed as means
± SE of four experiments.

Effects of AJ-9677 on white adipose tissues. The mice treated with
AJ-9677 showed an ∼50% reduction in the weight of epididymal white adipose
tissues compared with the control mice (Table
2). Moreover, triglyceride levels in the epididymal white adipose
tissues normalized to DNA levels (TG/DNA ratios) were also reduced by
∼50%, to almost the same level as the control levels
(Table 2), indicating that the
triglyceride content per cell was reduced to normal. TG/DNA ratios in inguinal
white adipose tissues were also reduced by ∼50% by AJ-9677 treatment
(Table 2).

Histological analysis of white adipose tissues. Treatment with
AJ-9677 clearly reduced the size of adipocytes in the epididymal white adipose
tissues (Fig. 3A and B. The number of
white adipocytes in the limited area was larger in the AJ-9677-treated group
than that in the control group. This effect was exerted in a dose-dependent
manner (i.e., a 120% increase at a dosage of 0.01 mg/kg and a 160% increase at
a dosage of 0.1 mg/kg) (Table 3).
Moreover, the AJ-9677 treatment caused the formation of some multilocular
adipocytes resembling brown adipocytes. Immunostaining of cytochrome oxidase
in the AJ-9677-treated group was stronger than that in the control group (Fig.
3C and D. These results were
also found in the inguinal white adipose tissues (data not shown). These
observations suggest that AJ-9677 treatment caused the exaltation of
mitochondrial function and the conversion of white adipocytes into brown
adipocytes. Alternatively, β3-adrenoceptor agonist treatment
may cause differentiation of preadipocytes of brown adipose tissue lineage in
white adipose tissues. AJ-9677 treatment caused a dramatic increase in the
number of mitochondria in the cytoplasm of inguinal white adipocytes (Fig.
3E and F).

Effect of AJ-9677 treatment on the overexpression of TNF-α and
leptin mRNA and protein. The TNF-α mRNA levels were markedly higher
in control KK-Ay/Ta mice than in C57BL/6J mice. Treatment
with AJ-9677 decreased the TNF-α mRNA level to 35% of the control level
(Fig. 4A). This
reduction of TNF-α mRNA was associated with a parallel reduction in the
amount of TNF-α protein secreted from the adipocytes
(Fig. 4C). Very
similar data were obtained for leptin mRNA and protein secretion
(Fig. 4B and
D). AJ-9677 also reduced the mRNA expression levels and
secretion of both TNF-α and leptin in the inguinal white adipose tissues
(data not shown).

Effect of AJ-9677 treatment on expression of UCP-1, -2,
and -3. AJ-9677 treatment caused the mRNA expression of UCP-1 in brown
adipose tissues to be increased threefold
(Fig. 5A). UCP-2 mRNA
levels were increased in the brown adipose tissues of control
KK-Ay/Ta mice, and AJ-9677 treatment further upregulated
the expression of UCP-2 mRNA. UCP-3 mRNA levels were not changed in the brown
adipose tissues of the control KK-Ay/Ta mice, and AJ-9677
treatment did not affect the expression of UCP-3 mRNA. UCP-1 was hardly
expressed in the epididymal and inguinal white adipose tissues of both control
KK-Ay/Ta and C57BL/6J mice. However, administration of
AJ-9677 caused a 20- to 80-fold increase in UCP-1 expression levels in the
epididymal (Fig. 5B)
and inguinal (Fig.
5C) white adipose tissues. UCP-2 expression levels were
increased three- to sixfold in both epididymal and inguinal white adipose
tissues of the control KK-Ay/Ta mice, as seen in brown
adipose tissues (Fig. 5B and
C). However, the expression of UCP-2 mRNA was reduced and
left unchanged by AJ-9677 in the epididymal and inguinal white adipose
tissues, respectively, unlike the effects seen in brown adipose tissues
(Fig. 5B and
C). The expression of UCP-3 was not altered in white and
brown adipose tissues of the control KK-Ay/Ta mice.
Although AJ-9677 treatment decreased UCP-3 expression levels in epididymal
white adipose tissues (Fig.
5B), it did not affect UCP-3 expression levels in
inguinal white adipose tissues (Fig.
5C). The expression levels of UCP-1, -2, and -3 mRNA in
the gastrocnemius were very low compared with the adipose tissues, and they
were not upregulated by AJ-9677 treatment
(Fig. 5D).

Messenger RNA expression and secretion of TNF-α and leptin in
epididymal white adipose tissues from control and AJ-9677-treated
KK-Ay/Ta diabetic obese mice and C57BL/6J mice. Adipose
tissues were removed after a 14-day treatment under well-fed conditions.
A and B: TNF-α and leptin mRNA expression,
respectively. The mRNA expression levels were analyzed by the RT-competitive
PCR method. C and D: TNF-α and leptin protein
secretion, respectively. TNF-α and leptin were measured by ELISA kits.
The details of the experiments are described in RESEARCH DESIGN AND METHODS.
The data are expressed as means ± SE; n = 8 in
KK-Ay/Ta groups and n = 5 in the C57BL/6J group.
**P < 0.01 and ***P < 0.001
compared with control KK-Ay/Ta group. N.D., not
detected.

Expression levels of UCP-1, -2, and -3 mRNA in adipose tissues and the
gastrocnemius from control and AJ-9677-treated KK-Ay/Ta
diabetic obese mice and C57BL/6J mice. Tissues were removed after a 14-day
treatment under well-fed conditions. A shows interscapular brown
adipose tissues; B shows epididymal white adipose tissues; C
shows inguinal white adipose tissues; and D shows the gastrocnemius.
The mRNA expression levels were analyzed by the RT-competitive PCR method. The
details of the experiments are described in RESEARCH DESIGN AND METHODS. Data
are expressed as means ± SE; n = 5-8 in
KK-Ay/Ta groups and n = 5 in the C57BL/6J group.
*P < 0.05, **P < 0.01, and
***P < 0.001 compared with the control
KK-Ay/Ta group. N.S., not significant compared with the
control KK-Ay/Ta group. #P < 0.05 compared
with the C57BL/6J group.

Protein expression levels of GLUT4 in epididymal white adipose tissues
from control and AJ-9677—treated KK-Ay/Ta diabetic
obese mice. Tissues were removed after a 14-day treatment under well-fed
conditions. Crude and plasma membrane fractions were prepared and analyzed by
Western blotting method. The detail was described in RESEARCH DESIGN AND
METHODS. Crude, crude membrane; PM, plasma membrane. □, control
KK-Ay/Ta mice; ▪, AJ-9677—treated
KK-Ay/Ta mice. The data are expressed as means ±
SE; n = 6 in each group. ***P < 0.001 compared
with control KK-Ay/Ta group.

DISCUSSION

Administration of AJ-9677 for 14 days reduced plasma glucose, insulin, FFA,
and triglyceride levels in the diabetic obese mouse model to almost the normal
levels seen in C57BL/6J mice. AJ-9677 stimulated lipolysis in adipocytes
dose-dependently in vitro. Although the plasma FFA level increased after the
first AJ-9677 administration, this effect was not observed after chronic
treatment, presumably because plasma FFAs generated by lipolysis may be
consumed by thermogenesis. Although we used the dosage of 0.1
mg-1·kg-1·day-1, AJ-9677 can
reduce these plasma parameters at lower dosages (data not shown). The
effective dosage was much lower for AJ-9677 than for other
β3-adrenoceptor agonists reported previously
(14,39).
White adipose tissues in humans show lower expression levels of
β3-adrenoceptor than in rodents. However, because AJ-9677 is
an equally potent agonist against both human and rat
β3-adrenoceptors in the Chinese hamster ovary cell expression
system, it may show the same kind of effects, to some extent, in humans as it
does in rodents.

Mitochondrial UCPs, which cause respiration without ATP synthesis, are
believed to be involved in the expenditure of excess energy. At least three
UCP isoforms (UCP-1, -2, and -3) have been cloned. UCP-1 is primarily
expressed in brown adipose tissues, and it is slightly expressed in white
adipose tissues. In the present study, administration of AJ-9677 increased the
expression of UCP-1 threefold in brown adipose tissues and 20- to 80-fold in
epididymal and inguinal white adipose tissues. The expression of UCP-2 was
increased by 2.5-fold in brown adipose tissues, but it was decreased in
epididymal white adipose tissues and unaltered in inguinal white adipose
tissues. The expression of UCP-3 was not changed in brown adipose tissues and
inguinal white adipose tissues, but it was decreased in epididymal white
adipose tissues. Previous reports showed that the
β3-adrenoceptor agonists BRL 35135 and CL 316,243
(40,41,42)
increased the expression of UCP-1 in both brown and white adipose tissues. Our
data are consistent with those results. The effect of AJ-9677 on the
expression of UCP-2 in brown adipose tissues is different from that of BRL
35135 (40), but consistent
with CL 316,243 (41). The
effects on UCP-3 expression are inconsistent. Thus, the effects of
β3-adrenoceptor agonists on UCP-2 and UCP-3 expression may
depend on the types of β3-adrenoceptor agonists, the duration
of the treatment, and the time of death. Although CL 316,243 increased UCP-1
mRNA in the gastrocnemius (7),
AJ-9677 did not increase UCP-1, 2, or 3 mRNA in the tissues. These differences
may also depend on the types of β3-adrenoceptor agonists. The
role of UCPs in the muscles may not be very important for the antidiabetic
effects of AJ-9677.

The major novel finding of this study is that the
β3-adrenoceptor agonist AJ-9677 reduced the size of white
adipocytes, an effect associated with the reduction of mRNA expression and
protein secretion of TNF-α and the reduction of plasma FFA levels. The
reduction in size of the white adipocytes was confirmed by histological
analysis and the triglyceride/DNA ratio. This reduction may be caused by
increased lipolysis and energy expenditure mediated by the increased
expression of both UCP-1 and UCP-2 after AJ-9677 treatment. We cannot identify
the source of UCP-1 overexpressed in white adipose tissues; it is possible
that the increased UCP-1 was expressed in the multilocular adipocytes found in
the white adipose tissues during AJ-9677 treatment. However, because the
absolute expression level of UCP-1 in white adipose tissues was much lower
than in brown adipose tissues, white adipose tissues may contribute less to
energy expenditure than brown adipose tissues. Thiazolidinediones cause the
differentiation of pre-adipocytes into adipocytes (through the peroxisome
proliferator—activated receptor γ [PPAR-γ]) to generate
small white adipocytes and, concomitantly, cause the apoptosis of large white
adipocytes (23). This
reduction of the mean size of adipocytes by thiazolidinediones is associated
with the normalization of both increased levels of expression of TNF-α
and increased production of FFAs in diabetic obese animal models. Because the
increase in the proportion of small adipocytes by thiazolidinediones appears
to contribute to the amelioration of insulin resistance via decreased
TNF-α expression and decreased FFA production, the reduction in the size
of adipocytes by AJ-9677 may contribute to the amelioration of insulin
resistance via a similar reduction of TNF-α expression and FFA
production. Moreover, the reduction in the size of adipocytes by AJ-9677 may
also be associated with decreased levels of leptin expression and secretion in
white adipose tissues, which may well reflect the abrogation of the need for
compensation for insulin resistance.

TNF-α can cause downregulation of GLUT4 expression
(43,44),
so decreased TNF-α expression probably contributes to increased GLUT4
expression after AJ-9677 treatment. Moreover, GLUT4 expression on the plasma
membrane was preferentially increased compared with the crude membrane,
suggesting that AJ-9677 treatment stimulated GLUT4 translocation to the plasma
membrane in white adipose tissues. Whether this effect is a direct action of
AJ-9677 or secondary to its amelioration of high plasma FFA levels is unclear
at present. In either case, the change in the localization of GLUT4 to the
plasma membrane, in addition to the increased GLUT4 expression levels, may
further contribute to the amelioration of insulin resistance and diabetes.

We would like to propose a novel hypothesis for the relationship between
the generation of small adipocytes and the amelioration of insulin resistance,
as depicted in Fig. 7. The
present study clearly shows that the β3-adrenoceptor agonist
AJ-9677 converted large adipocytes into small adipocytes through increased
lipolysis and the induction of UCPs (e.g., UCP-1) that are associated with the
reduction of TNF-α and FFA levels and thus the amelioration of insulin
resistance. We previously showed that thiazolidinediones promote adipocyte
differentiation to generate small adipocytes, which is also associated with
reduction of TNF-α and FFA levels, leading to the amelioration of
insulin resistance (23).
Therefore, insulin sensitivity is induced in vivo whether small adipocytes are
generated de novo by differentiation via stimulation of PPAR-γ or by
conversion from the large adipocytes via β3-adrenoceptors.
Thus, we would like to propose that the generation of small adipocytes is the
key event in the amelioration of insulin resistance.

Proposed mechanism of generation of small adipocytes and amelioration of
insulin resistance by β3-adrenoceptor agonist (AJ-9677).
AJ-9677 converts large adipocytes into small adipocytes through increased
lipolysis and the induction of uncoupling proteins (e.g., UCP-1) that are
associated with the reduction of TNF-α and FFA, which leads to the
amelioration of insulin resistance. Thiazolidinediones promote adipocyte
differentiation to generate small adipocytes that are also associated with the
reduction of TNF-α and FFA, which leads to the amelioration of insulin
resistance. Therefore, insulin sensitivity is induced in vivo whether small
adipocytes are generated de novo by differentiation via stimulation of
PPAR-γ or by conversion from the large adipocytes via
β3-adrenergic receptors. The generation of small adipocytes is
the key event in ameliorating insulin resistance.

Hypertrophic obesity resulting from adipocyte hypertrophy, which develops
with a high-fat diet and sedentary lifestyle, is closely linked to major
health issues (e.g., diabetes, hypertension, hyperlipidemia, and
cardiovascular diseases) in Western countries and in Japan
(45). Insulin resistance,
which is usually associated with hypertrophic obesity, is believed to be the
major mechanism causing these diseases. Thus, the treatment of hypertrophic
adipocytes is one of the most important issues in medical science.
Thiazolidinediones may not be the ideal agents because the number of
adipocytes tends to increase and promote obesity, even though insulin
resistance is ameliorated. This study clearly demonstrated that
β3-adrenoceptor agonists (e.g., AJ-9677) appear to convert
hypertrophic adipocytes into small adipocytes, thereby ameliorating insulin
resistance and obesity simultaneously.

Auboeuf D, Vidal H: The use of the reverse
transcription-competitive polymerase chain reaction to investigate the in vivo
regulation of gene expression in small tissue samples. Anal
Biochem245: 141
-148, 1997